It is known that each organ or tissue possesses a specific enzyme pattern which differs to a lesser or greater degree from the enzyme patterns of other organs and/or tissues and also from those of the surrounding body fluids. In case of cell damage or tissue damage, the enzyme patterns of the surrounding body fluids usually become changed in a manner specific for the diseased organ or tissue.
These findings have been utilized in enzyme diagnostics for some time, in order to draw conclusions from the changes of the enzyme pattern in the body fluids with respect to the damaged organs and/or tissues, or with respect to the type of the disease present therein. In accordance with the usual method of enzyme diagnostics, the test fluids obtained from body fluids, e.g., whole blood, plasma, serum and liquor, from tissues, e.g., blood corpuscles, liver, kidney, muscle and brain, and from excretions, e.g., urine, stool and sputum, are examined qualitatively and/or quantitatively for the enzymatic activities present therein.
However, the results of these examinations are equivocal to a substantial extent, since the proportions of enzyme activities in many organs and/or tissues are very similar or even identical. Therefore, a genuine organ specificity of the diagnosis can be achieved only in rare cases.
In this situation, the solution suggests itself to employ the finding that several enzymes occur in multiple molecular configurations and form so-called "isozymes" for diagnostic purposes. Isozymes are formed, for example, by aldolase, hexokinase, creatine kinase and lactate dehydrogenase and by alkaline and acidic phosphatase, various transaminases (aminotransferases) and dehydrogenases. The total activity of these enzymes is the sum of the activities of their various isozymes. Although isozymes catalyze the same reaction, they possess more or less different compositions and exhibit differing physical and biochemical properties.
The concentrations of the various isozymes of an enzyme are widely different in various organs or organ structures of the human body. In individual cases, the isozymes are even strictly organ-specific. Therefore, by the determination of the isozyme pattern in body fluids, tissues or excretions, a more extensive differential diagnosis can be accomplished than by the customary determination of the enzyme pattern. However, to do so requires a quantitative determination of the individual isozyme activities of an enzyme.
The catalytic properties of the isozymes are, in most cases, so similar that a differential determination of the isozyme activities by conventional methods of enzyme activity determination is impossible. In the past, various methods have been utilized in an attempt to differentiate between the isozymes of a diagnostically important enzyme:
1. differential heat inactivation; PA1 2. differential adsorption on sorption agents; PA1 3. differential activity determination with various substrates; and PA1 4. electrophoretic separation. PA1 1. The use of pure human isozyme antigens for the preparation of specific antisera; PA1 2. The use of practically completely precipitating antisera. PA1 3. The use of the entire isozyme pattern for the organ-specific and/or disease-specific diagnosis, insofar as the isozymes of the pattern are relevant for an organ specificity or disease specificity of the diagnosis. PA1 a. exhibit a different isozyme pattern in various human organs and tissues, within an organ or tissue, or within a cell; PA1 b. can be quantitatively precipitated immunologically; and PA1 c. are subject to differing genetic control.
The methods recited under (1) through (3) do not permit a complete differentiation of the isozyme activities, so that a quantitative determination of the isozyme pattern is impossible. The method set forth under (4) permits a complete separation of the isozymes only when the isolectric points thereof are far enough apart. In any event, the quantitative determination of the isozyme activities after the separation is time-consuming and error-prone.
Therefore, it would be desirable to have available a novel method by which the isozyme activities of the diagnostically important enzymes can be quantitatively determined as a matter of routine with an accuracy sufficient for diagnostics. Such a method would constitute a significant advance in the state of the organspecific and in the disease-specific differential diagnostic art.
A novel technique will be described hereinbelow which makes possible by simple means the determination of the various isozymes of an enzyme in a practically quantitative manner.
Methods have been described which make it possible to differentiate individual isozymes of several enzymes existing in multiple molecular forms by means of a combination of enzymological and immunological methods. According to these procedures, antisera are produced against more or less purified isozymes (as antigens). However, since these immune sera are responsive not only to the antigenic isozyme, but also to contaminations by other isozymes and other proteins in the sample to be tested, they do not contain the pure antibodies against the desired isozyme, but instead contain a multitude of further antibody activities which are in a more or less close relationship to the antibody activity against the one isozyme.
Additionally, antisera have been employed which had been obtained by the immunization with animal isozyme antigens. However, since animal and human isozymes possess a more or less differing molecular structure, the thus-obtained antisera against these isozymes also differ from one another. The antisera directed against animal isozymes are not absolutely specific with respect to the human isozymes, so that in diagnostic investigations on humans, it is impossible to obtain a quantitative impression of the isozyme pattern of an enzyme of interest.
In some of the conventional methods, it has been impossible to attain a flawless selective precipitation between isozyme (antigen) and the homologous antisera, so that inhibition of the enzymatic activity by the antigen-antibody complex had to be used for determining the antigen-antibody complex. However, in these inhibition methods, the antigen-antibody complex remains in the test serum. Such antigen-antibody complexes can also possess enzymatic activity. Thus, the immunosuppression normally cannot be rendered quantitative. Consequently, certain systematic errors, which are difficult to exclude and thus also difficult to estimate, are incurred in these determinations.
Attempts have also been made to cause the precipitation of the non-precipitable isozyme-antibody complexes by adding an excess of antiserum for complex formation with the isozyme (antigen) and then precipitating the entire complex, together with all other .gamma.-globulins, with anti-.gamma.-globulin sera. However, in contrast to the diagnostic method of this invention, this procedure requires a further quantitative predetermination of the titel of .gamma.-globulins, so that exactly equivalent amounts of anti-.gamma.-globulin can be added to the complex. Also, the reaction with the anti-.gamma.-globulin serum requires longer reaction times, e.g., approximately 12 hours, and thus is less suitable for the practice of routing diagnostics.
The methods known heretofore generally exhibited the disadvantage that they detected only a minor portion of the isozymes of an enzyme occurring in multiple molecular forms. For example, one could merely determine in practice that, in case of certain diseases, the total activity of an enzyme was more or less eliminated by one of the isozyme antisera, without taking into account the changes in the remaining isozymes. This type of diagnosis, therefore, remained more or less empirical. Only by an analysis of all isozyme activities of the enzyme of interest is it possible to render a faultless diagnosis based on organ specificity. Such an analysis is possible only when all isozymes, or all isozymes recognized as essential to the diagnosis of the enzyme of interest, have been obtained in the pure form from humans and the corresponding specific antisera thereagainst have been produced. By means of these separate antisera, it is then possible to flawlessly decipher the total activity present in the test serum of the individual isozymes.
The results heretofore evolved in this field of research lying between enzymology and immunology could heretofore be evaluated for diagnostic purposes only with difficulty. This was due, on the one hand, to the fact that the complicacy of the described methods were complicated which made it infeasible to employ them routinely and, on the other hand, the methods had too wide a margin of error, so that the results were scarcely usable for differential diagnostics.
By means of the present invention, a reduction of complicated scientific methods to a diagnostic procedure which is realistic for practical purposes is attained, which is urgently desirable. At the same time, diagnostic accuracy is substantially increased. This result was achieved by